sTREM2 is associated with amyloid‐related p‐tau increases and glucose hypermetabolism in Alzheimer's disease

Abstract Microglial activation occurs early in Alzheimer's disease (AD) and previous studies reported both detrimental and protective effects of microglia on AD progression. Here, we used CSF sTREM2 to investigate disease stage‐dependent drivers of microglial activation and to determine downstream consequences on AD progression. We included 402 patients with measures of earliest beta‐amyloid (CSF Aβ1‐42) and late‐stage fibrillary Aβ pathology (amyloid‐PET centiloid), as well as sTREM2, p‐tau181, and FDG‐PET. To determine disease stage, we stratified participants into early Aβ‐accumulators (Aβ CSF+/PET−; n = 70) or late Aβ‐accumulators (Aβ CSF+/PET+; n = 201) plus 131 controls. In early Aβ‐accumulators, higher centiloid was associated with cross‐sectional/longitudinal sTREM2 and p‐tau181 increases. Further, higher sTREM2 mediated the association between centiloid and cross‐sectional/longitudinal p‐tau181 increases and higher sTREM2 was associated with FDG‐PET hypermetabolism. In late Aβ‐accumulators, we found no association between centiloid and sTREM2 but a cross‐sectional association between higher sTREM2, higher p‐tau181 and glucose hypometabolism. Our findings suggest that a TREM2‐related microglial response follows earliest Aβ fibrillization, manifests in inflammatory glucose hypermetabolism and may facilitate subsequent p‐tau181 increases in earliest AD.

Thank you again for submitting your work to EMBO Molecular Medicine. We have heard back from three referees who agreed to evaluate your manuscript. As you will see from the reports below, the referees acknowledge the potential interest of the study. However, they raise a series of concerns, which we would ask you to address in a major revision of the manuscript. I think that the referees' recommendations are relatively straightforward, so there is no need to reiterate their comments. All issues raised by the referees need to be satisfactorily addressed. As you may already know, our editorial policy allows in principle a single round of major revision so it is essential to provide responses to the reviewers' comments that are as complete as possible. Please feel free to contact me in case you would like to discuss in further detail any of the issues raised by the reviewers.
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The statical analysis seems sometimes a bit of data stretch and does not follow the most commonly used methods. I would like to see if the results will remain doing more straightforward analysis of the FDG results.
Referee #2 (Remarks for Author): Biel and colleagues studied 402 ADNI participants stratified into early Aβ accumulators (CSF+/PET−) and late Aβ accumulators (CSF+/PET+) based on assumptions that the CSF Aβ abnormality occurs before PET Aβ. They reported that in the early Aβ accumulators, Aβ was associated with TREM2 and p-tau, and TREM2 mediated the association of Aβ and p-tau. Higher TREM2 was associated with glucose hypermetabolism. On the other hand, in late accumulators, Aβ was not associated with TREM2, but TREM2 was associated with p-tau and glucose hypometabolism. This is an interesting manuscript on an important topic which is the association between Aβ, tau phosphorylation, and glial markers. Results showing that TREM2 plays a role in the link between Aβ deposition and tau hyperphosphorylation are very relevant to the field and may have important implications for the biological definitions of the disease.
I have some concerns with the analysis -Is the significant interaction term reported to FDG also significant for p-tau?
-The manuscript's title suggests that TREM2 mediates amyloid-associated glucose hypermetabolism. It is not clear from the analyses the presence of mediation in glucose metabolism. Was there significant mediation with FDG? -The analysis in Figure 2B seems a bit of a stretch. The authors made associations in 2 groups anchored in controls and then compared T values of these associations from various regions. Are any of that T values significant after multiple comparisons in the first place? or the authors are comparing a bunch of non-significant t-value ? I would like to see the actual significant T-map after multiple comparison correction in each one of the groups. Can the authors compare t-values with the method they did statically speaking?
-The link between the results obtained with p-tau and FDG is not very clear and seems sometimes disconnected.
Referee #3 (Comments on Novelty/Model System for Author): These studies are excellently conducted and statistically analyzed and present incremental, yet important insight into the relationship of sTREM2 as a surrogate for microglial activation and the progression of AD.
Referee #3 (Remarks for Author): In this study Biel and coworkers describe the relationship between amyloid, sTREM2, and pTau 181 in early stage of amyloid and late stages of amyloid pathology. The authors find that higher levels of amyloid by PET were associated with higher sTREM2 and p-tau181, where as in late accumulators higher sTREM2 was associated with higher p-tau181 with no association with amyloid PET. Interestingly, sTREM2 was associated wither higher glucose metabolism in early accumulators, but lower in late accumulators. The relationship between metabolism, amyloid, tau, and neuroinflammation is important to understanding the disease process and possible timing of therapeutics targeting microglia. Additional analyses could increase the power of the study.
Major critques: 1.) Given the authors' previous work on the relationship between sTREM2 and APOE4 it would be interesting to include APOE4 as a covariate in the study to assess whether the effects of sTREM2 on ptau-181 and glucose metabolism were influenced by APOE genotype 2.) It would be interesting to extend the comparisons with other CSF p-tau epitopes (ie p-tau 217 or p-tau 231) if available to see if sTREM2 influences those phosphorylations as well.
We thank the editor and the reviewer's for their encouraging feedback on our manuscript. Below please find our replies including a description of all changes that were made to the manuscript according to the comments raised by the reviewer's. We hope that we have sufficiently addressed all concerns and believe that the manuscript has been improved and strengthened during the review process.
Please note that we merged two summary paragraphs in the result section (p. 11).
Referee #1 (Remarks for Author): In this study, Biel and colleagues have demonstrated that in people with early amyloid accumulation, that is those with abnormal CSF Aβ42 levels but normal Aβ-PET scans (Aβ CSF+/PET-), higher baseline Aβ-PET centiloid were associated with increased baseline and longitudinal levels of CSF soluble Triggering Receptor Expressed on Myeloid Cell 2 (sTREM2) and p-tau181. Furthermore, the authors report that in Aβ CSF+/PET-, the associations between Aβ-PET and cross-sectional as well as longitudinal CSF p-tau181 were mediated by sTREM2. In late Aβ-accumulators (Aβ CSF+/PET+), no associations were seen between Aβ-PET and sTREM2, whereas there was a positive association between baseline CSF levels of sTREM2 and p-tau181. Lastly, the author found opposite associations between sTREM2 and FDG-PET in early vs. late Aβ-accumulators; higher CSF levels of sTREM2 were associated with glucose hypermetabolism and hypometabolism in Aβ CSF+/PET-and Aβ CSF+/PET-, respectively. The manuscript is generally well-written and the results are clearly presented. The topic of the study is of high interest given that understanding of the relationship between TREM2 and Alzheimer's disease (AD)-related Aβ and tau pathologies is critical for design of therapeutic strategies targeting TREM2 to promote protective functions of microglia in AD. However, the lack of longitudinal imaging data is a limitation of the study and even though longitudinal CSF sTREM2 and p-tau181 data were available the samples size was small particularly in the early Aβ-accumulator group. This is my main concern especially considering a recent paper from DIAN that have highlighted the importance of longitudinal data when investigating the relationship between sTREM2 and AD pathological processes. Other issues that need to be addressed:

Reply:
We thank the reviewer for the thoughtful comments on our manuscript. We agree that the lack of longitudinal imaging data is a shortcoming of this study and further acknowledge this limitation in the discussion of the manuscript. p. 18: "Third, sample sizes were considerably small for early Aβ-accumulators when including longitudinal CSF sTREM2 (n=21) and CSF p-tau 181 (n=20) data, however, results were consistent for both cross-sectional and longitudinal associations. Further, we could only assess cross-sectional relationships between sTREM2 and FDG-PET. Here, it will be critical to assess longitudinal associations between sTREM2 and changes in FDG-PET once larger longitudinal datasets become available." 1. CFF Aβ42 was used to classify study participants as early or late Aβ-accumulators. However, the ratio of Aβ42 to Aβ40 would be a better measure because it is less affected by non-AD interindividual differences in total Aβ levels.

Reply:
We agree that a normalization of CSF Aβ using the Aβ 42 /Aβ 40 ratio would be the preferable measure. Unfortunately, Aβ 42 /Aβ 40 ratio data are only available on ADNI since 2019, and most of the sTREM2 assessments have been obtained before. Therefore, we could not generate sufficient data using this ratio in the current sample. We added this point in the limitations section.
25th Nov 2022 1st Authors' Response to Reviewers p. 17/18: "Second, we used CSF Aβ 1−42 levels for patients' classification, however, we recognize that a normalization of Aβ using the Aβ 42 /Aβ 40 ratio would be the preferable measure which should be applied once more data become available in ADNI or other datasets." 2. In statistical analysis, adjustment for multiples comparisons should be applied.

Reply:
In our first analysis testing the association between Centiloid, CSF sTREM2 and CSF p-tau 181 , different samples (early vs. late Aβ-accumulators) and different datasets (cross vs. longitudinal) were used testing different hypotheses. Hence, no correction for multiple comparisons was statistically needed, given that different hypotheses were addressed. For the assessment of the relationship between CSF sTREM2 and FDG-PET, the interaction term reached significance ( Fig.2A) and the surface rendering of t-values was an additional exploratory step (Fig.2B). We would like to point out that the latter analysis should underpin a different association pattern between sTREM2 and FDG-PET in early vs. late accumulators and not locate to specific ROIs that drive the relationship that was observed for the FDG-PET meta-ROI. We added a section in the methods to clarify the exploratory character of this analysis. p. 8: "For post-hoc exploration, we repeated the analysis in a brain wide manner across 200 ROIs that cover the entire neocortex (…). Note that the latter analysis was only exploratory to assess the general pattern between sTREM2 and FDG-PET in early vs. late Aβ-accumulators and thus not corrected for multiple comparisons."

Reply:
We would like to highlight that we have already displayed the association between sTREM2 and FDG-PET for both groups separately in Fig.2B. Therefore, we are assuming that the reviewer refers to a surface rendering of the interaction effect across the pooled sample (lm[each ROI ~ sTREM2 * Group + age + sex + education + diagnosis]). We added a respective figure in the Extended View. p. 13: "The brain surface projection of the T-values of the sTREM2 by group interaction on FDG-PET is made available in the Expanded View (Fig. EV1).". This figure will be made available online.

Reply:
We thank the reviewer for pointing out that the longitudinal association between sTREM2 and p-tau 181 was missing for early Aβ-accumulators. The cross-sectional association between sTREM2 and p-tau 181 was already included in the manuscript: "Further, higher sTREM2 levels were associated with higher p-tau 181 levels (β=0.587, T=5.425, p<0.001)." We added the missing longitudinal analysis. p. 10: "We obtained congruent results using longitudinal CSF data, (…). In addition, higher baseline sTREM2 levels were associated with faster subsequent change rates in p-tau 181 (β=0.938, T=6.286, p<0.001)."

Were there any associations between longitudinal changes in sTREM2 levels and longitudinal changes in p-tau181 or CSF Aβ levels.
Reply: To answer the reviewers question we tested associations between longitudinal changes in sTREM2 and longitudinal changes in p-tau 181 or CSF Aβ 1−42 . We found significant associations between changes in sTREM2 and changes in p-tau 181  Since we needed to generate an additional dataset including longitudinal CSF Aβ 1−42 for this analysis and are unsure whether this analysis will bring added value to the manuscript, we decided not to include it in the final version. However, it will be available online in the rebuttal in case the manuscript is accepted for publication.
6. The authors could consider focusing the introduction and the discussion specifically on the sTREM2 studies rather than on the role of microglia in AD.

Reply:
Several studies we referred to in the introduction or discussion were already using sTREM2 as a proxy for microglial activation. As in the papers themselves, we were using the expression "microglial activation" as a synonym for sTREM2. However, we clarified now in our manuscript which cited studies used sTREM2. p. 3: "Here, activation of microglia, the brains innate immune system, may play a key role in modulating these initial events in the amyloid cascade (10). The Triggering Receptor Expressed on Myeloid Cell 2 (TREM2) regulates the change of microglia from a homeostatic state to a disease associated state (11,12), and is a well-established in vivo proxy for microglial activation in . Yet, previous studies have yielded conflicting findings on a detrimental or protective role of microglial activation or TREM2-related microglial responses in AD." p. 3: "Similarly, studies in sporadic AD patients found that a TREM2-related microglial response is strongly correlated with soluble p-tau but not with Aβ levels (20) and that a TREM2-related microglial response may promote the development of aggregated tau pathology in AD, as measured via tau-PET (10, 21)." p. 4: "On the contrary, in symptomatic sporadic AD patients and patients with autosomal dominantly inherited AD, a higher TREM2-related microglial response has been associated with attenuated cognitive decline, amyloid accumulation and neurodegeneration (13, 28, 29)." p. 4: "Similarly, an animal model has shown that TREM2 loss of function is associated with increased Aβ seeding, (…)" p. 4: "In the present study, we used CSF soluble TREM2 (sTREM2) (…)" Although we used sTREM2 as a proxy of microglial activation, we think that referring to studies using different assessments of microglial activation (i.e. TSPO-PET, post-mortem, etc.) is important to gain a holistic picture of the involvement of microglia in the pathogenesis of AD. We already included a paragraph addressing this topic in the discussion. p. 16: "The different biomarkers of microglia (e.g. TSPO, CSF sTREM2) may reflect different aspects of the microglial response to the pathology. A current limitation is the lack of other microglial biomarkers that reflect the wide repertoire of microglia states. Therefore, we believe that it is crucial to better understand how microglia and sTREM2 are involved in the molecular progression of tau pathology, from tau hyperphosphorylation and increases in soluble p-tau to the development and spread of fibrillary tau pathology, in order to evaluate microglia and specifically TREM2-related pathways as a treatment target." 7. In the discussion, parts of the first paragraph and the beginning of the second paragraphs are repetitive.

Reply:
We agree with the reviewer and shortened the beginning of the second paragraph. p. 14: "Our first finding showed that the earliest sTREM2-related microglial response in AD is associated with fibrillar yet subthreshold Aβ increases (i.e. centiloids below 20), and that higher sTREM2 mediated the earliest Aβ-related increases in soluble p-tau 181 levels. In contrast, in participants showing fully developed Aβ pathology as indicated by combined positivity on both CSF and PET (i.e. late Aβ-accumulators), only associations between fibrillar Aβ (i.e. centiloid) and p-tau 181 or between p-tau 181 and sTREM2 were found." Referee #2 (Comments on Novelty/Model System for Author): The statical analysis seems sometimes a bit of data stretch and does not follow the most commonly used methods. I would like to see if the results will remain doing more straightforward analysis of the FDG results.

Referee #2 (Remarks for Author):
Biel and colleagues studied 402 ADNI participants stratified into early Aβ accumulators (CSF+/PET−) and late Aβ accumulators (CSF+/PET+) based on assumptions that the CSF Aβ abnormality occurs before PET Aβ. They reported that in the early Aβ accumulators, Aβ was associated with TREM2 and p-tau, and TREM2 mediated the association of Aβ and p-tau. Higher TREM2 was associated with glucose hypermetabolism. On the other hand, in late accumulators, Aβ was not associated with TREM2, but TREM2 was associated with p-tau and glucose hypometabolism. This is an interesting manuscript on an important topic which is the association between Aβ, tau phosphorylation, and glial markers. Results showing that TREM2 plays a role in the link between Aβ deposition and tau hyperphosphorylation are very relevant to the field and may have important implications for the biological definitions of the disease.

Reply:
We appreciate the reviewers' reasoned comments and suggestions for improving our manuscript. We hope we have sufficiently addressed the remaining concerns.

I have some concerns with the analysis -Is the significant interaction term reported to FDG also significant for p-tau?
Reply: This is an interesting question, however, the interaction term for an sTREM2 by group interaction on p-tau levels did not reach significance (lm[p-tau 181~ sTREM2 *Group + age + sex + education + clinical status]. We included this analysis in the methods and results. p. 8: "Lastly, we combined early and late Aβ-accumulators and tested for an sTREM2 by group interaction on cross-sectional and longitudinal p-tau 181 levels, controlling for age, sex, education, and clinical status." p. 12: "No sTREM2 by group interaction on CSF p-tau 181 levels were found when pooling early and late T=0.145,p=0.885;p=0.731)." This suggests that sTREM2 is associated with higher p-tau levels across the AD continuum, but that glucose metabolism is differently associated with sTREM2. This is in line with previous work, showing that activated microglia consume a majority of the FDG-PET tracer (Xiang et al., Sci Transl Med, 2021), which may result in the observed hypermetabolism when neurodegeneration and resulting hypometabolic brain changes are not yet apparent. Please note also, that we have discussed this in the paper (p. 16+17).
-The manuscript's title suggests that TREM2 mediates amyloid-associated glucose hypermetabolism. It is not clear from the analyses the presence of mediation in glucose metabolism. Was there significant mediation with FDG?

Reply:
We agree with the reviewer that we did not explicitly test a mediation effect for the association between sTREM2 and amyloid-associated glucose hypermetabolism. Therefore, we appreciate the comment that the title can be misleading and changed it into "sTREM2 is associated with amyloid-related p-tau increases and glucose hypermetabolism in Alzheimer's" -The analysis in Figure 2B

seems a bit of a stretch. The authors made associations in 2 groups anchored in controls and then compared T values of these associations from various regions. Are any of that T values significant after multiple comparisons in the first place? or the authors are comparing a bunch of non-significant t-value ? I would like to see the actual significant T-map after multiple comparison correction in each one of the groups. Can the authors compare t-values with the method they did statically speaking?
Reply: For the assessment of the relationship between CSF sTREM2 and FDG-PET, our main analyses focused on the interaction term of group (i.e. early vs. late Aβ-accumulators) by sTREM2 on the FDG-PET meta-ROI (i.e. a summary ROI of AD-vulnerable brain regions) reached significance ( Fig.2A). This was the significant main analysis on which we base our conclusion that sTREM2 relates to increased metabolism in early Aβ-accumulators vs. decreased metabolism in late Aβ-accumulators. The surface rendering of T-values was an additional exploratory step (Fig.2B), in which we aimed to examine whether the association between sTREM2 and hypo/hypermetabolism was generally apparent across the brain or restricted to particular brain regions. We would like to point out that the latter analysis should underpin a different overall association pattern of sTREM2 with FDG-PET between early and late Aβ-accumulators and not locate specific ROIs with a significant interaction effect. Note also, that we tested main effects of sTREM2 and not interactions with group here. We added a section in the methods to clarify the exploratory character of this analysis. Therefore, the T-values can rather be seen as an effect size measure that indicates the directionality of an association between sTREM2 and FDG-PET in the early vs. late accumulators. p. 8: "For post-hoc exploration, we repeated the analysis in a brain wide manner across 200 ROIs that cover the entire neocortex (…). Note that the latter analysis was only exploratory to assess the general pattern between sTREM2 and FDG-PET in early vs. late Aβ-accumulators and thus not corrected for multiple comparisons." In addition to the t-test we added a non-parametrical comparison using confidence intervals (CI), which provide non parametric support that associations between sTREM2 and FDG-PET show an overall opposing directionality in early vs. late Aβ-accumulators. : "In addition, 95% CIs of the 200 T-values did not overlap (early Aβ-accumulators: CI 1.086-1.249; late Aβ-accumulators: CI −1.302-−1.098) (…)."

p. 8+9: "For non-parametric comparison, we determined 95% confidence intervals (CI) for the 200 T-values for early and late
Please note also that reviewer 1 raised a similar concern and asked us to repeat the sTREM2 by group interaction analyses on a group level, and show the full T-map, which is shown in our response to comment 3 by reviewer 1.
-The link between the results obtained with p-tau and FDG is not very clear and seems sometimes disconnected.

Reply:
We understand the reviewers concern and added an exploratory section in the limitation. The rationale of including FDG-PET was that we have previously shown that p-tau precedes tau aggregates (Pichet Binette et al., Nat Comm, 2022), which in turn ensue neurodegeneration. Since not sufficient tau-PET data were available, we could not directly assess the effect of sTREM2 on the association between p-tau and tau-PET or tau-PET and neurodegeneration. Therefore, we only assessed the link between sTREM2 (which we showed to be associated with subsequent p-tau increases) and neurodegeneration (i.e. FDG-PET). However, including tau-PET to the equation will be an important step for future studies. p. 18: "Fourth, we could not assess p-tau 181 by sTREM2 interactions on tau aggregates since tau-PET data were limited and thus not sufficient to reliably assess relationships with sTREM2 in the current dataset. Here, it would be of interest to assess the association between tau aggregates, sTREM2, and FDG-PET. Although we observed group differences (early vs. late Aβ-accumulators) for the association between sTREM2 and FDG-PET, the current study cannot disentangle whether sTREM2 plays a moderating role for the association between tau-PET and FDG-PET. (…)." Referee #3 (Comments on Novelty/Model System for Author): These studies are excellently conducted and statistically analyzed and present incremental, yet important insight into the relationship of sTREM2 as a surrogate for microglial activation and the progression of AD.
Repl y: We thank the reviewer for the overall positive feedback on our manuscript and the helpful suggestions for improvements.

Referee #3 (Remarks for Author):
In this study Biel and coworkers describe the relationship between amyloid, sTREM2, and pTau 181 in early stage of amyloid and late stages of amyloid pathology. The authors find that higher levels of amyloid by PET were associated with higher sTREM2 and p-tau181, where as in late accumulators higher sTREM2 was associated with higher p-tau181 with no association with amyloid PET. Interestingly, sTREM2 was associated wither higher glucose metabolism in early accumulators, but lower in late accumulators. The relationship between metabolism, amyloid, tau, and neuroinflammation is important to understanding the disease process and possible timing of therapeutics targeting microglia. Additional analyses could increase the power of the study.

Reply:
We repeated the main analyses including APOE4 status and added the results in the Expanded View. p. 9: "Finally, the main analyses were repeated including APOE4 status as a covariate to assess whether APOE4 influences the effect of sTREM2 on p-tau 181 or glucose metabolism. Subjects were classified as APOE4 risk allele carriers when at least one ε4 allele was detected." p. 11: "All analyses remained consistent when including APOE4 genotype as a covariate (except for the cross-sectional ACME in early Aβ-accumulators, which only reached borderline significance (p=0.058; Table EV1+EV2))." p. 13: "The analyses remained consistent when including APOE4 genotype as a covariate (β=−0.379, T=−1.972, p=0.050)." Thank you for the submission of your revised manuscript to EMBO Molecular Medicine. We have received the enclosed reports from the three referees who agreed to re-assess it. As you will see, while Reviewers #2 and #3 are now supportive, Reviewer #1 still raised some concerns about the generalizability of the findings and requested replication in an independent cohort. We would ask you to discuss this raised point as potential limitations and future directions in writing.
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Sincerely, Jingyi
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